Delayed diagnoses of diseases are a major concern in hospitals. For example, a delayed diagnosis of multi-drug resistant bacteria (MDRB) may lead to heightened mortality and morbidity because these bacteria are highly contagious and the rate of infection of these bacteria exponentially increases in a matter of hours. The treatment and hospitalization of patients due to, delayed diagnoses also pose a huge economic burden to the healthcare industry. Furthermore, centralized hospitals, in particular, face huge logistic and economic burden where typically more than 300 patients are screened daily for pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) pathogens.
For example, MRSA infections cost the US healthcare system in excess of US$20 billion dollars annually. The problem is further exacerbated by a rapidly increasing rate of MRSA infections, accounting for 60% of Staphylococcus aureus infections in 2004 as compared to 22% in 1995. In Singapore's public hospitals, patients infected with MRSA bacteria are 10 times more likely to die during hospitalization as compared to uninfected patients. These patients also stayed 4.6 times longer in hospital and faced four times higher hospital-related costs.
Apart from saving lives, rapid detection of pathogens enables healthcare staff to undertake mitigating measures in a timely manner, such as quarantining patients and determining an effective treatment regime.
Current phenotypic methods of diagnosis are slow, ranging from 18-24 hours or even longer for certain diseases, for example about 4-6 weeks for tuberculosis. This is because such phenotypic methods of diagnosis, such as antimicrobial susceptibility testing for accurate detection of MRSA, are very much dependent on the growth rate of bacterial culture. Thus, these methods typically take as long as 1-4 days, during which time the rate of infection and patient mortality would increase significantly. Therefore, the existing phenotypic approach needs to be complemented with a method for detecting resistant genes, such as polymerase chain reaction (PCR) or real-time PCR. Even though the presence of a target-resistant gene may not confer phenotypic resistance and novel resistant genes will not be detected, PCR is undoubtedly a rapid and sensitive assay for detecting known target-resistant genes.
However, PCR has to be conducted in a highly multiplexed manner, given the sheer number of resistant genes implicated in any given bacterial species. For example, in the case of MRSA infections, healthcare providers are not only interested in the presence of the principal resistant gene, which is mecA, but in addition to that, they would like to ascertain the presence of bacterial and species-specific control genes, such as 16SrRNA and nuc, as well as other antibiotic-resistant genes, such as ermA, blaZ and msrA. In fact, there are well over 20 target genes of interest for MRSA infections alone.
Administering the right treatment is also as critical a task as detecting the presence of pathogens. For example, conventional wisdom suggests the use of broad-spectrum antibiotics for treatment of MRSA infections, in the absence of an antibiotic resistance profile. However, there are disadvantages to the use of broad-spectrum antibiotics. Furthermore, new treatments, such as the use of bacteriophages, require further studies to show that they work in an in vivo setting. As for now, narrow spectrum antibiotics are widely viewed as a viable treatment option for MRSA infections provided the patient is rapidly screened for all relevant antibiotic-resistant genes using PCR.
In the screening of antibiotic-resistant genes using PCR, performing multiple singleplexed PCR reactions is not a viable option for two reasons. Firstly, this significantly increases the number of PCR reactions per patient, and therefore limits the number of patient samples that can be processed simultaneously. Secondly, the sensitivity of the assay is also adversely affected given that patient sample, which is limited to a single nasal swab with no culturing step involved, has to be split over several reactions. Further, as mentioned above, there are multiple target genes to be screened. On the other hand, a multiplexed PCR would address these concerns since multiple antibiotic-resistant target genes will be amplified in a single PCR reaction, effectively increasing patient throughput by multiple folds.
Real-time PCR enables multiplexed detection. However, the number of target genes detected is low, typically ranging from 1 to 3, and in some cases, 4 to 5 targets. The low multiplexing is mainly due to the limitation in the number of fluorescence-conjugated DNA probes that are optically separable. Consequently, the emission bandwidth is effectively limited to 500-700 nm. Furthermore, there is significant overlap between the excitation and emission spectra of organic fluorophores, typically attached at the 5′ end of the DNA probes.
Alternatively, instead of real-time PCR, end-point multiplexed PCR assays may also be a viable option since only the presence or absence of resistance genes are needed to be determined.
Multiplexed PCR incorporates multiple primer pairs in a single reaction where each primer pair amplifies a certain target gene. Gene amplification is then followed by an end-point detection assay, such as gel electrophoresis and melt-curve analysis, using a DNA binding dye to confirm the presence of the target gene. Target genes are usually detected based on the size (gel electrophoresis) or melting temperature (melt-curve analysis) of the respective amplicons. However, such detection methods have limited specificity since amplicons of two different target genes may have similar size and/or melt temperature, or alternatively, the sizes and melt temperatures may be too close in value such that the amplicons may not be accurately resolved. Such scenarios are highly likely in a highly multiplexed assay whereby a large number of target genes are detected. Also, gel electrophoresis is highly time-consuming and labor-intensive.
DNA microarray is seen as a viable end-point detection assay where multiplexed PCR is first performed to generate single-stranded amplicons, followed by hybridization of amplicons to sequence-specific probes immobilized on a chip. However, the workflow is time-consuming and labor-intensive, whereby the chip is incubated with the PCR product for several hours, followed by a series of washing steps. Surface treatment of the chip is also required to immobilize the probes onto the chip surface and to ensure DNA localization at the probe spots during incubation. The high equipment cost is another concern since robotics technology is used for spotting probes onto the chip surface, and the optical setup incorporates a scanner for transitioning from one field of view to another so as to cover the entire chip area.
DNA sequencing is another platform technology that enables sequencing of the genome and analysis to determine if known mutations or resistant determinants are present. However, DNA sequencing is still a time-consuming process that may take several days, and there is also a compromise between sequencing speed and sequencing errors that may arise. DNA purification is a key precursor to DNA sequencing, but it is also a limiting factor for rapid diagnosis of bacterial infection and antibiotic resistance since overnight culturing is first performed to obtain pure species-specific bacterial colonies from which DNA is extracted.
Lastly, newer sequencing technologies may substantially reduce sequencing time, but the costs associated with purchasing these machines and running the assays remain high.
There is therefore a need to provide a device and system that overcomes, or at least ameliorates, one or more of the disadvantages described above. There is a need for a rapid, high-throughput and accurate method of detecting target molecules.